The methylotrophic yeast Pichia pastoris is by far the most often used yeast species in the production of recombinant proteins and is used in thousands of laboratories worldwide for the production of proteins for fundamental studies, as drug targets and for therapeutic use, and as a model for peroxisomal proliferation and methanol assimilation. The P. pastoris expression technology is available from Invitrogen (Carlsbad, Calif.) and from Research Corporation Technologies (Tucson, Ariz.), making it accessible for academic and commercial purposes alike. P. pastoris grows to high cell density, provides tightly controlled methanol-inducible transgene expression and efficiently secretes heterologous proteins in defined media. Indeed, several P. pastoris-produced biopharmaceuticals that are either not glycosylated (such as human serum albumin) or for which glycosylation is only needed for proper folding (such as several vaccines) are already on the market. P. pastoris strains with small, homogenous N-glycans have also been generated, which were then further engineered into human-type N-glycosylation1,2. Glyco-engineered products are now moving to clinical development3. Moreover, monoclonal antibodies can be made at gram per liter scale in glycosylation-homogeneous strains4. For further strain engineering, a better understanding of all aspects of the yeast's protein production machinery is desired, and a number of studies relating to Pichia's secretory system and engineered promoters have been forthcoming5,6. However, although P. pastoris is widely used for protein production, relatively few genetic tools, engineered strains and data on the biology of this organism are available.